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Intravital Imaging of Dynamic Bone and Immune Systems Methods and Protocols 1st Edition Masaru Ishii (Eds.)
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Preface
During the last decade, advanced intravital imaging technology by using multiphoton excitation fluorescent microscopy has revolutionized the field of biological sciences. Based on these techniques, we are now able to visualize in situ behavior of a diversity of living cells “intravitally” within intact tissues and organs. This research trend would be quite meritorious especially for analyzing bone and immune systems, where various kinds of cell types are moving around and their spatiotemporal control is pivotal for proper functions in vivo. For example, bone is a mysterious organ where various kinds of hematopoietic and immune cells are produced and functioning although poorly analyzed by conventional methodology such as histological analyses with decalcified bones. Intravital imaging of bones has identified the behavior of bone cells such as osteoclasts, specialized macrophages contributing to bone destruction, revealing novel mechanisms controlling their migration and function in situ. Furthermore, visualization of the dynamic movement of various cell types in lymph nodes, skin, kidney, nervous systems, and cancer tissues has identified crucial mechanisms and factors triggering their migration and infiltration in these areas.
Despite the increased importance of the methods in biological sciences and the availability of expensive imaging modalities such as multiphoton microscopy in many research institutes, one of the big hurdles preventing popularization of this research trend has so far been the complicated experimental protocol. Researchers need the procedures to be elaborated for themselves in their respective laboratories.
In this book, leading researchers who are actually doing imaging studies in the field of bone and immune systems contributed the chapters where they described respective actual cutting-edge protocols, including some “secret recipes.” These detailed methods would surely be useful for general readers in order to establish and perform these experiments on their own.
I express my sincere gratitude to all the authors for their willingness to share their secrets and to Prof. John Walker at Humana Press for giving me the opportunity to publish this book for the series. Both he and the authors have been patient during the editing of this volume.
Osaka, Japan
Masaru Ishii
15 Imaging Window Device for Subcutaneous Implantation Tumor ........................ 153
Wataru Ikeda, Ken Sasai, and Tsuyoshi Akagi
16 New Tools for Imaging of Immune Systems: Visualization of Cell Cycle, Cell Death, and Cell Movement by Using the Mice Lines Expressing Fucci, SCAT3.1, and Kaede and KikGR .................... 165 Michio Tomura
Contributors
Tsuyoshi AkAgi • KAN Research Institute Inc�, Kobe, Hyogo, Japan
Clemens AlT • Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
mArC BAjénoff • Aix-Marseille University, Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Centre d’Immunologie de Marseille-Luminy (CIML), Marseille, France
Pierre C. DAgher • Division of Nephrology, Department of Medicine, Indiana University, Indianapolis, IN, USA
gyohei egAwA • Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan
BriAirA geiger • Department of Chemistry, Richard Stockton College of New Jersey, Galloway, NJ, USA
reBeCCA genTek • Aix-Marseille University, Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Centre d’Immunologie de Marseille-Luminy (CIML), Marseille, France
ClémenT ghigo • Aix-Marseille University, Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Centre d’Immunologie de Marseille-Luminy (CIML), Marseille, France
Chi Ching goh • Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Singapore, Singapore; Department of Microbiology, Immunology Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
TAkAshi hATo • Department of Medicine, Indiana University, Indianapolis, IN, USA
TeTsuyA honDA • Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan
mATTeo iAnnACone • Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy; Experimental Imaging Center, IRCCS San Raffaele Scientific Institute, Milan, Italy
wATAru ikeDA • KAN Research Institute Inc , Kobe, Hyogo, Japan
mAsAru ishii • Department of Immunology and Cell Biology, Graduate School of Medicine, Osaka University, Osaka, Japan
yookyung jung • Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; Center for Molecular Spectroscopy and Dynamics, Institute for Basic Science (IBS), Seoul, Republic of Korea
kenji kABAshimA • Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan
TomoyA kATAkAi • Department of Immunology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
nAoTo kAwAkAmi • Institute of Clinical Neuroimmunology, University Hospital and Biomedical Center, Ludwig-Maximilians University Munich, Munich, Germany; Max-Planck Institute of Neurobiology, Martinsried, Germany
juniChi kikuTA • Department of Immunology and Cell Biology, Graduate School of Medicine, Osaka University, Osaka, Japan
mirelA kukA • Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy
jACkson liAngyAo li • Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Singapore, Singapore; School of Biological Sciences, Nanyang Technological University, Singapore, Singapore; CNIC (Fundación Centro Nacional de Investigaciones Cardiovasculares), Madrid, Spain
ChArles P. lin • Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
sAyAkA mATsumoTo • Department of Immunology and Cell Biology, Graduate School of Medicine, Osaka University, Osaka, Japan
ryohei mATsuurA • Department of Cardiovascular Surgery, Osaka University, Graduate School of Medicine, Osaka, Japan
shigeru miyAgAwA • Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan
mAsAyuki miyAsAkA • MediCity Research Laboratory, University of Turku, Turku, Finland; Interdisciplinary Program for Biomedical Sciences, Institute of Academic Initiatives, Osaka University, Suita, Osaka, Japan
lAi guAn ng • Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Singapore, Singapore; School of Biological Sciences, Nanyang Technological University, Singapore, Singapore; Department of Microbiology, Immunology Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
sAToshi nishimurA • Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan
AnThony P. rAPhAel • Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; Dermatology Research Centre, Translational Research Institute, School of Medicine, The University of Queensland, St Lucia, QLD, Australia
AnDreA reBolDi • Department of Pathology, University of Massachusetts Medical School, Worcester, MA, USA
juDiTh r. runnels • Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
AsukA sAkATA • Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan
sTefAno sAmmiCheli • Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy
ken sAsAi • KAN Research Institute Inc , Kobe, Hyogo, Japan
yoshiki sAwA • Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan
Contributors
joel A. sPenCer • Center for Systems Biology and Wellman Center for Photomedicine, Center for Regenerative Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; School of Engineering, University of California Merced, Merced, CA, USA
AkirA TAkeDA • MediCity Research Laboratory, University of Turku, Turku, Finland
miChio TomurA • Laboratory of Immunology, Faculty of Pharmacy, Osaka Ohtani University, Osaka, Japan
eiji umemoTo • Laboratory of Immune Regulation, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
seTh winfree • Department of Medicine, Indiana University, Indianapolis, IN, USA
juwell w. wu • Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
Chapter 1
Bone Imaging: Osteoclast and Osteoblast Dynamics
Junichi Kikuta and Masaru Ishii
Abstract
Bone is continually remodeled by bone-resorbing osteoclasts and bone-forming osteoblasts. Although it has long been believed that bone homeostasis is tightly regulated by communication between osteoclasts and osteoblasts, the fundamental process and dynamics have remained elusive. To resolve this, we established an intravital bone imaging system using multiphoton microscopy to visualize mature osteoclasts and osteoblasts in living bone.
We herein describe the methodology for visualizing the in vivo behavior of bone-resorbing osteoclasts and bone-forming osteoblasts in living bone tissues using intravital multiphoton microscopy. This approach facilitates investigation of cellular dynamics in the pathogenesis of bone-destructive disorders, such as osteoporosis and rheumatoid arthritis in vivo, and would thus be useful for evaluating the efficacy of novel anti-bone-resorptive drugs.
Key words Intravital imaging, Multiphoton microscopy, Osteoclast, Osteoblast, pH-sensing probe
1 Introduction
Bone is a dynamic tissue that undergoes continuous remodeling by bone-resorbing osteoclasts and bone-forming osteoblasts [1]. Tight control of bone remodeling is critical for maintaining bone homeostasis in response to structural and metabolic demands. Bone remodeling is strictly controlled through a complex communication network between osteoblast- and osteoclast-lineage cells [2]. Therefore, it is essential to understand the spatial–temporal relationship and interactions between osteoblasts and osteoclasts in vivo. In particular, it remains controversial whether these cell types physically interact with each other.
Bone is the hardest tissue in the body; for this reason, it is technically difficult to visualize cellular interactions in the bone marrow cavities of living animals. The morphology and structure of bone tissues can be analyzed using various conventional methods, including micro-computed tomography, histomorphological analyses, and flow cytometry. These methods yield information on cell
shape and gene expression patterns, but not on dynamic cell movements in living bone marrow. The recent introduction of fluorescence microscopy has enabled imaging of the cellular dynamics of organs and tissues in vivo [3, 4]. Therefore, we established an advanced imaging system to visualize living bone tissues using intravital multiphoton microscopy [5–8]. For visualization of deep bone tissue, we selected the parietal bone of mice, which is ~80–120 μm thick (within the range of infrared lasers), as the observation site. In this region, living bone marrow can be accessed with minimal invasion.
To visualize mature osteoclasts (mOCs), we generated transgenic reporter mice expressing tdTomato, a red fluorescent protein, in the cytosol of mOCs (TRAP-tdTomato mice) [8]. To visualize mature osteoblasts (mOBs), we also generated fluorescent reporter mice expressing enhanced cyan fluorescent protein (ECFP) in the cytosol of mOBs (Col2.3-ECFP mice). To visualize communication between mOCs and mOBs, we crossed TRAPtdTomato mice with Col2.3-ECFP mice to generate TRAPtdTomato/Col2.3-ECFP double fluorescently labeled mice. Using intravital multiphoton microscopy of calvaria bone tissues of TRAP-tdTomato/Col2.3-ECFP mice, we successfully visualized the in vivo behavior of living mOCs and mOBs on the bone surface; the imaging results suggested direct interactions between mOCs and mOBs in vivo. In wide views of skull bones under normal conditions, mOCs and mOBs appeared to be distributed separately, although some direct, albeit spatiotemporally limited, mOC–mOB interactions were evident. Analysis of time-lapse images showed that mOCs and mOBs exhibited distinct spatial distributions. However, several mOCs in contact with mOBs displayed dendritic shapes and projected synapse-like structures toward mOBs. Additionally, these interactions between mOCs and mOBs changed dynamically according to bone homeostatic conditions.
We recently developed pH-sensing chemical fluorescent probes to detect localized acidification by bone-resorbing osteoclasts on the bone surface in vivo [9, 10]. These probes are based on the boron-dipyrromethene (BODIPY) dye and a bisphosphonate group. BODIPY dyes have a large number of applications because of their environmental stability, large molar absorption coefficients, and high fluorescence quantum yields [11]. The bisphosphonate group replaces phosphate ions in hydroxyapatite, the main component of bone tissue, and forms a tight bond with the bone matrix. Therefore, the bisphosphonate group facilitates delivery and fixation of the probe on bone in living animals [12]. Our recently developed pH-probes enabled visualization of bone resorption by osteoclasts, which led to identification of two distinct functional states of differentiated osteoclasts: bone-resorptive [R] and nonresorptive [N].
2 Materials
2.1 Multiphoton Microscopy
2.1.1 Standard Imaging
2.1.2 Spectral Imaging (See Note 5)
2.2 Mice and Anesthesia
2.3 Intravital Imaging
Bone Imaging: Osteoclast and Osteoblast Dynamics
In this chapter, we describe a methodology for visualizing the in vivo behavior of osteoclasts and osteoblasts simultaneously in living bone tissue. In addition, we describe imaging of osteoclast function using a pH-sensing fluorescent chemical probe.
1. Upright multiphoton microscope (A1R-MP; Nikon) (see Note 1).
2. Water-immersion objective, 25× (APO: numerical aperture [NA], 1.1; working distance [WD], 2.0 mm; Nikon) (see Note 2).
3. Femtosecond-pulsed infrared laser (Chameleon Vision II Ti: sapphire laser; coherent) (see Note 3).
4. External non-descanned detector (NDD) with four channels (Nikon).
5. Dichroic and filter set: Three dichroic mirrors (458, 506, and 561 nm), and four band-pass filters (417/60 nm for the second harmonic generation (SHG) signal, 480/40 nm for ECFP, 534/30 nm for autofluorescence, and 612/69 nm for tdTomato) (Nikon) (see Note 4).
6. NIS Elements integrated software (Nikon).
1. Upright multiphoton microscope (LSM 780 NLO; Carl Zeiss).
2. Water-immersion objective, 20× (W Plan-Apochromat: NA, 1.0; WD, 2.4 mm; Carl Zeiss).
1. Custom-made stereotactic stage (Fig. 1) (see Note 6).
2. Head holder with a hexagonal window (see Note 7).
3. Shaver and hair-removal lotion.
4. Iris scissors and tweezers for mouse surgery.
5. N-Butyl cyanoacrylate glue.
Junichi Kikuta and Masaru Ishii
Fig. 1 Schematic illustration of calvaria bone imaging. The mouse is anesthetized with isoflurane, the frontoparietal region of the skull bone is surgically exposed, and its head is immobilized using the custom-made stereotactic holder. The head holder is kept fully loaded with PBS by an infusion syringe pump
11. Environmental dark box in which an anesthetized mouse is warmed to 37 °C by an air heater.
2.4 Preparation of the pH-Sensing Probe
2.5 Staining of Blood Vessels
2.6 Image Processing and Analysis
1. pH-sensing chemical fluorescent probe (Fig. 2) [10].
2. PBS immersion buffer, pH 7.4.
3. One 26-gauge needle.
1. Angiographic agent: Qtracker 655.
2. One 29-gauge insulin syringes for intravenous injection.
1. Image processing and analysis software: Imaris (Bitplane), NIS Elements (Nikon), and ZEN (Carl Zeiss).
2. After Effects software (Adobe).
Fig. 2 Chemical structure of the pH-sensing fluorescent chemical probe. The pH-sensing probe is based on the boron-dipyrromethene (BODIPY) dye for visualization in low-pH environments in bone created by active osteoclasts; the bisphosphonate group facilitates delivery of the probe to bone tissue. Fluorescence of the probe can be detectable under acidic condition
3 Methods
3.1 Administration of the pH-Sensing Probe
3.2 Intravital Multiphoton Imaging of Calvaria Bone
1. Dissolve 5 mg/kg of pH-sensing chemical probe (pHocas-3) in PBS.
2. All procedures in mice are performed under anesthesia.
3. Inject the pH-probe subcutaneously into mice daily beginning 3 days prior to imaging.
4. Perform intravital bone imaging experiments (see Subheading 3.2).
1. Start up the multiphoton microscope and turn on the heater in the environmental dark box (see Note 8).
2. All surgical procedures are performed under isoflurane inhalation anesthesia.
3. Shave the hair and apply hair-removal lotion to the top of the head of the mouse (see Note 9).
4. Disinfect the skin of the head using 70% ethanol.
5. Cut the skin minimally using iris scissors and then expose the frontoparietal region of the skull.
6. Resect marginal muscles and the periosteum.
7. Apply n-butyl cyanoacrylate glue to the back of the head holder.
8. Apply ethyl-cyanoacrylate glue to the marginal area of the exposed bone, but not to the skull bone within the hexagonal window.
Junichi Kikuta and Masaru Ishii
9. Place the head holder on the skull bone using the skull bone suture as an anatomical landmark.
10. Wait a few minutes to allow the glue to firmly secure the skull bone to the head holder (see Note 10).
11. Fix the head holder to the custom-made stereotactic stage using two screws, and immobilize the mouse as tightly as possible to avoid drift secondary to respiration and pulsation (Fig. 1) (see Note 11).
12. Set the infusion line in the groove of the head holder, and fill the hexagonal window with PBS (see Note 12).
13. Place the mouse in the environmental dark box.
14. If necessary, intravenously inject Qtracker 655 dissolved in PBS.
15. Focus on the bone marrow cavity at an appropriate depth and look through the ocular lenses using a mercury lamp as the light source.
16. Change the light source from the mercury lamp to the Ti: sapphire laser.
17. Set the excitation wavelength, zoom ratio, z-positions, interval time, and duration time using the microscope software (see Note 13).
18. Observe the bone tissue by multiphoton excitation microscopy.
19. Monitor the heart rate of the mouse using an electrocardiogram throughout imaging (see Note 14).
3.3 Image Processing and Analysis
3.3.1 Spectral Unmixing for Images Acquired by 32-Channel GaAsP Spectral Detectors
3.3.2 Analysis of Intravital Multiphoton Images
1. Obtain the SHG, autofluorescence, pHocas-3, ECFP, and tdTomato fluorescence spectra from the raw images using ZEN software by manually selecting appropriate pixels.
2. Utilize these spectral libraries for spectral unmixing algorithms.
3. Discriminate each fluorescence signal, exclude autofluorescence, and create unmixed images (Fig. 3).
1. Correct images for XY drift using NIS Elements or Imaris software.
2. Analyze images by measuring the frequency and duration of cell-to-cell contact using Imaris software (Fig. 4).
3. Create the movie using After Effects software.
4 Notes
1. There are two types of microscope: upright and inverted. Bone marrow can be observed using an inverted microscope. Multiphoton microscopes are also available from other manufacturers (e.g., Leica Microsystems and Olympus).
Fig. 3 Visualization of the bone-resorptive function of mature osteoclasts using a pH-sensing fluorescent probe. Representative images of the calvaria of TRAPtdTomato mice treated with a pH-probe, showing sites of local bone resorption. Red, mature osteoclasts expressing TRAP-tdTomato; green, fluorescence signals from areas of high H+ concentration; blue, bone tissue. Arrowheads and asterisks represent bone-resorptive and non-resorptive osteoclasts, respectively. Scale bar, 40 μm
Fig. 4 Visualization of living mature osteoclasts and osteoblasts on bone surface by intravital multiphoton microscopy. Representative image of the calvaria of TRAP-tdTomato/Col2.3-enhanced cyan fluorescent protein (ECFP) mice under control conditions. Red, mature osteoclasts (mOCs) expressing TRAP-tdTomato; cyan, mature osteoblasts (mOBs) expressing Col2.3-ECFP. Arrowheads represent mOB–mOC interactions. Scale bar, 40 μm
Junichi Kikuta and Masaru Ishii
2. Objective lenses with a higher NA and longer WD are desirable.
3. A femtosecond-pulsed infrared laser is also available from Spectra-Physics (MaiTai).
4. The dichroic and filter set required depends on the fluorescent proteins and dyes used.
5. We utilize a Carl Zeiss upright multiphoton microscope with internal 32-channel GaAsP spectral detectors when using simultaneous multiple fluorescent labels. This enables acquisition of the entire spectrum of each label in one scan, which is then used to create unmixed images.
6. The custom-made stereotactic stage is composed of a 10-mmthick metal plate, on which two cylinders with screw holes are placed. The head holder can be fixed to this stage with two stainless screws.
7. The head holder has one recess, the curvature radius of which is 28 mm. The center of the recess has a hexagonal window. The head holder is 3 mm in thickness and 15 g in weight.
8. Some time is required for the laser and temperature to stabilize.
9. Remove as much hair as possible to prevent hair (which is autofluorescent) entering the visual field.
10. Prevent glue from contaminating the visual field because some glues are autofluorescent.
11. Do not fasten too tightly to avoid injuring the mouse.
12. The head holder is kept fully loaded with PBS by an infusion syringe pump.
13. The excitation wavelength of 940 nm is used to simultaneously excite ECFP, tdTomato, and pHocas-3. For an example of intravital time-lapse bone imaging, image stacks were collected at 3 μm vertical steps at a depth of 50–150 μm below the skull bone surface with 2.0× zoom, 512 × 512 X–Y resolution, and a time resolution of 5 min.
14. The heart rate is used as a guide to adjust the anesthetic gas concentration.
References
1. Hattner R, Epker BN, Frost HM (1965) Suggested sequential mode of control of changes in cell behavior in adult bone remodeling. Nature 206:489–490
2. Sims NA, Martin TJ (2014) Coupling the activities of bone formation and resorption: a
multitude of signals within the basic multicellular unit. Bonekey Rep 3:481
3. Cahalan MD, Parker I, Wei SH, Miller MJ (2002) Two-photon tissue imaging: seeing the immune system in a fresh light. Nat Rev Immunol 2:872–880
4. Germain RN, Miller MJ, Dustin ML, Nussenzweig MC (2006) Dynamic imaging of the immune system: progress, pitfalls and promise. Nat Rev Immunol 6:497–507
5. Ishii M et al (2009) Sphingosine-1-phosphate mobilizes osteoclast precursors and regulates bone homeostasis. Nature 458:524–528
6. Ishii M, Kikuta J, Shimazu Y, MeierSchellersheim M, Germain RN (2010) Chemorepulsion by blood S1P regulates osteoclast precursor mobilization and bone remodeling in vivo. J Exp Med 207:2793–2798
7. Kikuta J et al (2013) Sphingosine-1-phosphatemediated osteoclast precursor monocyte migration is a critical point of control in the anti-bone-resorptive action of active vitamin D. Proc Natl Acad Sci U S A 110:7009–7013
8. Kikuta J et al (2013) Dynamic visualization of RANKL- and Th17-mediated osteoclast function. J Clin Invest 123:866–873
9. Kowada T et al (2011) In vivo fluorescence imaging of bone-resorbing osteoclasts. J Am Chem Soc 33:17772–17776
10. Maeda H et al (2016) Real-time intravital imaging of pH variation associated with osteoclast activity. Nat Chem Biol 12:579–585
11. Loudet A, Burgess K (2007) BODIPY dyes and their derivatives: syntheses and spectroscopic properties. Chem Rev 107:4891–4932
12. Kozloff KM, Volakis LI, Marini JC, Caird MS (2010) Near-infrared fluorescent probe traces bisphosphonate delivery and retention in vivo J Bone Miner Res 25:1748–1758
Chapter 2
Intravital Imaging of Mouse Bone Marrow: Hemodynamics and Vascular Permeability
Yookyung Jung, Joel A. Spencer, Anthony P. Raphael, Juwell W. Wu, Clemens Alt, Judith R. Runnels, Briaira Geiger, and Charles P. Lin
Abstract
The bone marrow is a unique microenvironment where blood cells are produced and released into the circulation. At the top of the blood cell lineage are the hematopoietic stem cells (HSC), which are thought to reside in close association with the bone marrow vascular endothelial cells (Morrison and Scadden, Nature 505:327–334, 2014). Recent efforts at characterizing the HSC niche have prompted us to make close examinations of two distinct types of blood vessel in the bone marrow, the arteriolar vessels originating from arteries and sinusoidal vessels connected to veins. We found the two vessel types to exhibit different vascular permeabilites, hemodynamics, cell trafficking behaviors, and oxygen content (Itkin et al., Nature 532:323–328, 2016; Spencer et al., Nature 508:269–273, 2014). Here, we describe a method to quantitatively measure the permeability and hemodynamics of arterioles and sinusoids in murine calvarial bone marrow using intravital microscopy.
Key words Bone marrow blood vessel, Arterioles, Sinusoids, Permeability, Hemodynamics, Flow speed, Blood vessel diameter, Mouse restraint, Intravital imaging
1 Introduction
The microanatomic landscape of the bone marrow is dominated by the presence of a dense vascular network. The vasculature makes up approximately 25–30% of the marrow space by volume [1, 2], with sinusoidal blood vessels being the most prominent vessel type. However, the bone marrow vasculature is heterogeneous [3, 4], and the heterogeneity is particularly notable when imaging the local hemodynamics in real time [1, 5]. Compared to arteriolar vessels, the flow speed and sheer rate in sinusoidal vessels are much lower [1, 5], and vascular permeability is significantly higher [5]. Increased vascular permeability has been shown to increase the level of reactive oxygen species (ROS) in the surrounding cells and
Yookyung Jung and Joel A. Spencer contributed equally to this work.
to impact the migration and differentiation of hematopoietic stem and progenitor cells (HSPCs) [5]. Here, we present a protocol for measuring hemodynamics and permeability of arterioles and sinusoids in mouse bone marrow using video rate laser scanning confocal/two-photon microscopy [6–8]. Video rate laser scanning is achieved by a polygon-based fast scanning mechanism that allows full-frame (500 × 500 pixels) image acquisition at 30 frames per second. Higher frame rates can be acquired at a reduced frame size (120 frames per second at 500 × 125 pixels). The rapid scanning enables individual hemodynamic and vessel permeability measurements based on fluorescently labeled circulating blood cells and vascular dye leakage, respectively. Using this protocol, we could measure flow speeds up to 10 mm/s in individual blood vessels and simultaneously record vascular permeability in the same vessels within the first minute after dye injection. We also present concurrent methods for stabilizing the mouse for imaging and injection of cells and dyes on stage using a custom-built catheter.
2 Materials
2.1 Mouse
2.2 Fluorescence Labeling of Red Blood Cells (RBCs)
Any strain of mice can be used. Care should be taken to minimize signal overlap with the vascular dyes for permeability studies if coinjecting fluorescently labeled cells or using transgenic fluorescent mouse models. We typically use C57BL/6, which is the most common background strain for our studies. Animals were maintained in the animal facilities of Massachusetts General Hospital in compliance with institutional guidelines, and all animal studies were approved by the Subcommittee on Research Animal Care of the institution.
The high resolution imaging demands minimal mouse movement during imaging caused by breathing and other movement artifacts (see Note 1). Adequate mouse restraint can be achieved by modifying a 50 mL conical tube as described in [4]. However, with improved access to rapid-prototyping and related CAD software, more precise restraints can be fabricated. Our custom-built, 3D printed mouse restraint was designed using Autodesk® Inventor Professional 3D CAD software (Autodesk®, USA) and printed on a Fortus 380mc (Stratasys, USA) using ASA filament (see Note 2).
Fluorescently labeled RBCs are used for flow speed measurement. Here we assumed that the local blood flow speed is equal to the average speed of RBCs in the vasculature. Solutions and heparin-coated microcentrifuge tubes should be prepared immediately before RBC labeling.
1. Fluorescent cell label CFDA-SE: thaw one vial of 500 μg CFDA-SE (V12883, Invitrogen) and solubilize in 90 μL DMSO.
Yookyung Jung et al.
2.3 Fluorescence
Labeling of the Vasculature
2.4 Custom-Built Catheter
Hemodynamics and Permeability of Bone Marrow Blood Vessels
2. Labeling solution A (“Soln A”): 90 mL of PBS (without calcium and magnesium) supplemented with 1 g/L glucose and 0.1% bovine serum albumin (BSA). Keep 60 mL at room temperature, warm 30 mL to 37 °C.
3. Labeling solution B (“Soln B”): 48 mL PBS (without calcium and magnesium), warm to 37 °C.
4. Wash solution (“Soln W”): 30 mL PBS (without calcium and magnesium) supplemented with 1 g/L glucose. Keep at room temperature.
5. Donor mouse: we label 1.2 × 109 RBCs for a final yield of >0.75 × 109 cells. In our experience, a single facebleed from an adult unanesthetized C57BL/6 mouse provides sufficient RBCs.
6. Microcentrifuge tube for blood collection: add 200 μL 100 unit/mL heparin into tube and vortex briefly to coat.
7. Any BSA stock solution (concentration varies by manufacturer).
Vascular dyes are used for flow speed measurement, vessel diameter measurement, and vascular permeability measurement.
2. Tygon microbore tubing, 0.010 (ID) × 0.030 (OD) in.
3. ½ cc insulin syringe with 29 G × ½ in. needle.
3 Methods
3.1 Preparing
Fluorescently Labeled RBCs
RBCs are fluorescently labeled ex vivo and injected into recipient mouse 2–3 days before imaging to allow time for the bone marrow to re-establish its equilibrium hemodynamic state.
1. Perform facial vein blood collection on donor mouse and collect the blood in heparin-coated microcentrifuge tube. Animal anesthesia (if used) should be non-intravenous, such as isoflurane. Avoid ketamine and xylazine.
2. Transfer the heparinized blood suspension into 12 mL of Soln A at room temperature. Centrifuge at 500 × g for 6 min. Discard the suspension, which should be opaque with a faint red shade. The cell pellet should be bright red.
3. Wash the RBCs two more times with 1.5 mL Soln A at room temperature.
4. Perform cell count. Resuspend 1.2 × 109 RBCs in 12 mL Soln A (37 °C) at 1 × 108 cells/mL. Keep the cells at 37 °C.
5. Add the 90 μL CFDA-SE DMSO stock into Soln B. Vortex to mix and split the solution evenly into two 50 mL centrifuge tubes (24 mL each).
6. Mix 6 mL of the RBC suspension from step 4 into each of the two 50 mL centrifuge tubes prepared in step 5. Label at 37 °C for 12 min.
7. When labeling is complete, introduce bovine serum albumin stock solution such that BSA concentration is 0.1% in each of the two 50 mL centrifuge tubes. Centrifuge.
8. Wash each cell pellet in 30 mL Soln A at room temperature. Centrifuge.
9. Resuspend each cell pellet in 15 mL Soln W at room temperature and combine the two suspensions into one centrifuge tube. Perform cell count and centrifuge.
10. Resuspend the labeled RBCs in 180 μL saline for retro-orbital or tail vein injection. The RBC suspension should retain the same bright red hue as in blood; do not inject cells that look “rusty” as they will be cleared by circulation.
1. Anesthesia is induced using 4% isoflurane in oxygen with a maintenance rate of 1–3%. Induction is achieved in a separate chamber prior to mounting the mouse in the restraint.
2. For minimal motion, the mouse is first restrained by the bitebar, followed by the nose cone and lastly the side skull restraints. The contact points on the side of the skull should be between the eye socket and ears, typically in the region of the squamosal bones (see Fig. 1).
3. Following appropriate restraint, the skull is prepared after scalp incision [7].
The protocol for performing intravital optical imaging of the bone marrow has been well described [6–8]. It is summarized as follows. Figure 2a shows the cross-sectional view of the calvarial bone of a mouse. As the bone overlying the marrow cavity is typically 50–70 μm thick, the bone marrow can be accessed optically after a simple skin flap surgery to expose the underlying bone surface. The thickness of the marrow cavity can vary from tens of micrometers to a couple of hundred micrometers (Fig. 2a and b), where the low intensity region in between the bone layers is the bone marrow. Using the intrinsic second harmonic signal to visualize
3.2 Mouse Restraint and Preparation
3.3 Intravital Imaging of Calvarial Bone Marrow Yookyung
3.4 Imaging Labeled RBC and Calculating Flow Speed
Fig. 1 3D printed mouse holder for intravital optical imaging. (a) Mouse holder. Red arrows indicate the nose cone and bite-bar which are anchor points for securing the mouse head. Red dots are prongs that fix squamosal bones of the mouse. (b) Side view of mouse skull. Red arrows and dots of (a) are shown at the corresponding sites [6]
the bone, we image the bone marrow cavities located in the central region of the skull around the sagittal and coronal sutures, which encompass an area approximately 3–6 by 6–8 mm (see Fig. 2c and Note 3). An example image of a bone marrow cavity of a nestinGFP mouse is shown in Fig. 2d, with the bone signal in blue, GFP in green, hematopoietic stem and progenitor cell in red, and blood vessels in grey. The cell is overlaid on the pre-acquired nestin-GFP, blood vessels, and bone images.
The blood flow speed is measured by tracking the frame-to-frame displacement of individual RBCs using high frame rate imaging. 750–800 million fluorescently labeled RBCs are injected into a recipient mouse 2–3 days before imaging. Immediately prior to imaging, a fluorescent vascular dye (e.g., Rhodamine-B-dextran) is injected allowing the visualization of the blood vessels that map the “paths” of RBC displacement. For accurate flow speed measurement, the average velocity of a minimum of five RBCs is used. The detailed protocol for determining RBC and blood flow speed in vivo is as follows:
Fig. 2 Intravital optical imaging of the bone marrow through the calvarial bone. (a) Cross-sectional view of a mouse skull (from Henkelman M, Micro-CT images of mouse skull. http://www.stlfinder.com/model/mouseskull-(from-micro-ct)) and optical access into the bone marrow between calvarial bone layers. (b) Intensity profile of the dotted yellow line in (a) shows two layers of calvarial bone and bone marrow between them. (c) Top view of a mouse skull. Optical imaging area is shown as a dotted red rectangle that contains coronal and sagittal sutures [6]. (d) Example of in vivo optical imaging of the bone marrow [9]. Nestin-GFP (green), hematopoietic stem and progenitor cell (red), blood vessels (grey), and bone (blue) are displayed. The cell is overlaid on the pre-acquired nestin-GFP, blood vessels, and bone images. Scale bar is 50 μm
1. Deliver 40 μL of 10 mg/mL 70 kDa Rhodamine B-dextran (ThermoFisher Scientific) to a labeled RBC recipient mouse by retro-orbital or tail vein intravenous (i.v.) injection for vascular labeling.
2. Perform simultaneous in vivo imaging of bone, blood vessels, and labeled RBC. Imaging conditions are as follows:
● Image size: 500 × 125 pixels.
● Imaging speed: 120 frames/s.
● Bone: 840 nm femtosecond laser excitation, ~40 mW of power at the sample, 80 MHz repetition rate, MaiTai,
Yookyung Jung et al.
Hemodynamics and Permeability of Bone Marrow Blood Vessels
of labeled
in the bone marrow
Blue: second harmonic generation signal from
blood vessels. Green: labeled RBCs. Scale bars are 50 μm. (a) Example image showing overlay of labeled RBCs at several consecutive frames. (b) Flow of a single RBC in an arteriole. (c) Flow of a single RBC in a sinusoid
Spectra Physics, Collection of the second harmonic generation from collagen in bone.
● CFSE-labeled RBC: 491 nm continuous wave (CW) laser excitation, ~1 mW of power at the sample, Dual Calypso, Cobolt, Collection of confocal fluorescence at the spectral range of 509–547 nm.
● Blood vessel: 561 nm CW laser excitation, Jive, Cobolt, ~1 mW of power at the sample, Collection of confocal fluorescence at the spectral range of 573–613 nm.
3. Calculate the speed of the blood flow using the following equation (see Note 4):
Total distance traveled by RBC/Time (= number of frames × 1/120 s).
The flow of labeled RBCs in the bone marrow blood vessels is shown (Fig. 3). The labeled RBCs at several consecutive frames are overlaid on the vascular image. Dotted and solid arrows show the direction of flow and the positions of RBCs at each frame, respectively.
Arterioles with small diameters (5–10 μm) show fast flow (>1.5 mm/s) (Fig. 3b), and sinusoids with large diameter (>20 μm) show slow flow (<1 mm/s) (Fig. 3c).
Based on the flow speed and vessel diameter (Fig. 4), the majority of bone marrow blood vessels can be categorized into two distinct groups (Fig. 4f), those with high flow speed and small diameter
3.5 Blood Vessel Diameter Measurement
Fig. 3 Flow
RBCs
blood vessels.
bone collagen. Red:
Fig. 4 Flow speed and diameter of arterioles and sinusoids. (a) Two-photon image of the blood vessels in the calvarial bone marrow. Numbers in red and blue indicate the blood vessel identification (ID). Scale bar is 50 μm. (b, c) Individual blood vessel flow speeds from arterioles and sinusoids, respectively. (d, e) Individual blood vessel diameters from arterioles and sinusoids, respectively. (f) Scatterplot of flow speed as a function of diameter. Two distinct groups of “high flow speed and small diameter” vs “low flow speed and large diameter” can be found (data from 70 blood vessels, n = 6 mice). Specific site of the individual blood vessels with ID numbers in (b–e) can be found in (a)
(arterioles) vs those with low flow speed and large diameter (sinusoids). These results are consistent with published values [1]. The protocol for blood vessel diameter measurement is as follows:
1. Deliver 40 μL of 10 mg/mL 70 kDa Rhodamine B-dextran (ThermoFisher Scientific) by retro-orbital or tail vein intravenous (i.v.) injection for vascular labeling.
2. Acquire two-photon excitation fluorescence (at the spectral range of 550–680 nm) image of the vessels in the calvarial bone marrow immediately after injecting the vascular dye with femto-
3.6 Preparation of Custom-Built Catheter for Vascular Imaging and Permeability Measurement
3.6.1 Catheter Preparation
Hemodynamics and Permeability of Bone Marrow Blood Vessels
second laser source (~40 mW of power at the sample, 80 MHz repetition rate, 840 nm, MaiTai, Spectra Physics). Example image shown in Fig. 4a.
3. Measure the diameter of the blood vessels using image analysis software, for example ImageJ or Fiji software [10]. Repeated measurements are taken and the averaged value of the measured diameters is acquired.
Because of the high permeability of the bone marrow vasculature, it is necessary to perform dye injection while the mouse is on stage, so that the early dynamics of dye leakage immediately after injection can be recorded and analyzed.
1. Cut 30½ gauge needle by using forceps (Fig. 5(1)).
2. Insert the cut needle into 0.030 in. flexible plastic tube (Fig. 5(2)).
3. Insert a needle of an insulin syringe into the other end of the plastic tube (Fig. 5(3)).
Fig. 5 Preparation of home-built catheter for repeated vascular imaging and permeability measurement
Yookyung Jung et al.
4. Repeated bone marrow vascular imaging and permeability measurements can be performed by using the home-built catheter.
5. In case multiple measurements of permeability are necessary, vasculature dyes with distinct two-photon emission spectrum should be used to prevent the signal leaking between the detection channels (see Note 5).
3.6.2 Quantitative Measurement of Permeability
1. Perform video rate two-photon imaging of bone marrow vasculature immediately after injection of vascular dye.
2. Frame-to-frame misalignment of the images can exist because of residual movement artifacts from breathing (Fig. 6a).
3. Series of raw images in Fig. 6a are registered and aligned by either using Template Matching plugin of ImageJ or Matlab code utilizing the command, normxcorr2, which performs normalized two-dimensional cross-correlation [11, 12]. Ten
Fig. 6 Process to measure the rate of vascular dye leakage. (a) Series of two-photon raw vascular images with 1/30 s of time interval. Misalignment of the images exists because of residual movement artifacts. (b) Raw images are registered and aligned. Ten consecutive frames are frame-averaged to reduce the background noise signal. (c) Regions to measure the dye leakage are displayed as yellow rectangles labeled with 1 (next to sinusoid) and 2 (next to arteriole). (d) Average intensity in the region 1 and 2 of (c) is plotted as a function of time. The linear fits are shown as red lines. The slope of this linear fit is the rate of vascular dye leakage, dI/dt
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ornament; but the main figures and general design have perished. The walls of the opposite chamber were never cased with marble, so that the pilgrims were able to leave here the same tokens of their visits as they left at St. Sixtus’. The graffiti are of the same general character, but of a somewhat later date; the old forms of prayer have disappeared; most of the names and inscriptions are in Latin; and among the few that are Greek, there are symptoms of Byzantine peculiarities.
The chief object of interest, however, now remaining in these chambers is the epitaph which stands in the middle of the smaller room. Of course, this was not its original position; but it has been so placed, in order that we may see both sides of the stone without difficulty, for both are inscribed. The stone was originally used for an inscription in honour of Caracalla, belonging to the year 214. The Christian inscription on the other side professes to have been set up by “Damasus, Bishop, to Eusebius, Bishop and Martyr,” and to have been written by Furius Dionysius Filocalus, “a worshipper (cultor) and lover of Pope Damasus.” But it is easy to see at a glance that it never was really executed by the same hand to which we are indebted for so many other beautiful productions of that Pope. At first, therefore, and whilst only a few fragments of this inscription had been recovered, De Rossi was tempted to conjecture that it might be one of the earliest efforts of the artist who subsequently attained such perfection. At length, however, the difficulty was solved in a more sure and satisfactory way. A diligent search in the earth with which the chamber was filled brought to light several fragments of the original stone, on which the letters are executed with the same faultlessness as on the other specimens of its class. The visitor to the Catacombs may see them painted, in a different colour from the rest, in the copy of the epitaph which De Rossi has caused to be affixed to the wall; and he will observe that amongst them are some letters which are wanting in the more ancient copy transcribed on the reverse of Caracalla’s monument. It is clear that the original must have been broken in pieces, by the Lombards or other ancient plunderers of the Catacombs, and that the copy which we now see is one of the restorations by Pope Vigilus or some other Pontiff about that time (page 47). The copyist was so ignorant that he could only
transcribe the letters which were on the spot before his eyes, and, even when he was conscious that a letter was missing, he could only leave a vacant space, being doubtful how it should be supplied. Witness the space left for the first letter of Domino in the penultimate line of the inscription, and the word in altogether omitted in the third line.
“Heraclius forbad those who had fallen away [in times of persecution] to grieve for their sins. But Eusebius taught those unhappy men to weep for their crimes. The people are divided into parties; fury increases; Sedition, murder, fighting, quarrelling, and strife. Presently both [the Pope and the heretic] are exiled by the cruelty of the tyrant, Although the Pope was preserving the bonds of peace inviolate. He bore his exile with joy, looking to the Lord as his Judge.
And on the shore of Sicily gave up the world and his life.”
Having sufficiently considered the form of the inscription, let us now say a few words about its substance, which is important, because it restores to us a lost chapter of Church history. Every student knows how keenly contested in the early ages of the Church was the question as to the discipline to be observed towards those Christians who relapsed into an outward profession of Paganism under the pressure of persecution. There were some who would fain close the door of reconciliation altogether against these unhappy men (miseri), whilst others claimed for them restitution of all Christian privileges before they had brought forth worthy fruits of penance.
The question arose whenever a persecution followed after a long term of peace; for during such a time men’s minds were specially apt to decline from primitive fervour, and the number of the lapsed to increase. We are not surprised, therefore, to find the question agitated during the persecution of Decius in the middle of the third century. There is still extant a touching letter, written to St. Cyprian by the clergy of Rome at a time when the Holy See was vacant after the martyrdom of St. Fabian, which clearly defines the tradition and practice of the Church. In it they say that absolution was freely given to those of the lapsed who are in danger of death, but to others only when wholesome penance has been exacted; and they declare that “they have left nothing undone that the perverse may not boast of their being too easy, nor the true penitents accuse them of inflexible cruelty.” The same question arose under the same circumstances in the persecution of Diocletian. Pope Marcellus was firm in upholding the Church’s discipline, but he was resisted with such violence that public order was disturbed in the city by the strife of contending factions, and the Pope was banished by order of the Emperor Maxentius. This we learn from another inscription of Pope Damasus, who says that he wrote it in order that the faithful might not be ignorant of the merit of the holy Pontiff. Eusebius was the immediate successor of Marcellus, and the epitaph now before us is clearly a continuation of the same history, ending in the same punishment of the Pope, as the reward of his contention for the liberties of the
Church. For it should be remembered that these Popes were driven from their see and died in exile, not because they refused to apostatize, but because they insisted on maintaining the integrity of ecclesiastical discipline. They may justly be reckoned, therefore, among the earliest of that noble army of martyrs, who, from those days even to our own, have braved every danger rather than consent to govern the Church in accordance with other than the Church’s rules.
It yet remains to make two further remarks upon the epitaph of Pope Eusebius before we leave it. The first is, that he is called a martyr, though it nowhere appears that he really shed his blood; but this is by no means the only instance in which the title of martyr is given in ancient documents to men who have suffered for the faith and died whilst those sufferings continued. And secondly, it is to be observed that although we have no record of the translation of the body of St. Eusebius from Sicily to Rome, there is no reason to doubt the fact. All the earliest monuments speak of him as buried in a crypt of the Cemetery of St. Callixtus, and although the law forbad the translation of the bodies of those who had died in exile unless the emperor’s permission had been previously obtained, the old lawyers tell us that this permission was freely given. Numerous examples teach us the great anxiety of the ancient churches to have their bishops buried in the midst of them; no doubt, therefore, the necessary permission was asked for, as soon as a change in the imperial policy towards the Church made it possible; and the body of St. Eusebius was recovered and brought to Rome soon after his death, just as that of one of his predecessors, St. Pontian, had been brought from Sardinia by St. Fabian.
CHAPTER IV.
THE TOMB OF ST. CORNELIUS.
We have not promised to conduct the visitor to everything that is worth seeing in this cemetery, but only to enumerate and explain the principal monuments of historical importance which every stranger usually sees. And the only specimen of this class which remains to be spoken of is the tomb of St. Cornelius, which lies some way off. In order to reach it we must traverse a vast network of galleries, narrow and irregular, connecting what were once independent cemeteries, or at least were areæ added at various times to the Cemetery of Callixtus. If our guide is not in too great haste, he may allow us to step aside into two or three chambers by the way, in which are certain objects of interest worth looking at. The first is a long inscription belonging to the last decade of the third century, in which the Deacon Severus records that he has obtained leave from the Pope Marcellinus to make a double chamber, with arcosolia and a luminare, in which himself and his family may have quiet graves (mansionem in pace quietam). This is in the third area of the cemetery, next to the area in which we visited the crypt of St. Eusebius.
In the adjoining area, and belonging probably to the same date, is a very curious fresco, much damaged by having been cut through for the sake of making a grave behind it, yet still easily distinguishable in all its main features. The Good Shepherd occupies the centre of the painting. On either side is an apostle, probably SS. Peter and Paul, hastening away from Christ, Who has sent them to go and teach all nations. These are represented by two sheep standing before each of the apostles; and over their heads hangs a rock, whence pour down streams of water, which the apostles are receiving in their hands and turning on the heads of the sheep. We need no special explanation of this; we have already learnt that the Rock is Christ, and that the waters represent all Christian graces and sacraments. But what is worth noticing in this picture is the various attitudes of the sheep, and the corresponding distribution of the water. A perfect
torrent is falling on the animal that stands with outstretched neck and head uplifted, drinking in all he hears with simplicity and eagerness; whilst another, which has turned its back upon the apostle, is left without any water at all. Of the other two, one is standing with head downcast, as if in doubt and perplexity, and upon him too grace is still being poured out more abundantly than upon the fourth, which is eating grass, i.e., occupied with the affairs of this world.
On the right hand side of this arcosolium are two representations of Moses; in the one he is striking the rock, and one of the Jews is catching some of the water which gushes forth; in the other he is taking off his shoes, preparing to obey the summons of God, who is represented by a hand coming forth from the cloud. The painting on the other side of the arcosolium is even more defaced than that in the centre. A large semi-circular recess has been cut through it, and then the smoke of the lamp which burnt in this recess during the fourth and fifth centuries has almost obliterated the little that remained of the figure of our Lord. He stood between two of His apostles, who are offering Him bread and fish, and six baskets of loaves stand on the ground before them.
And now we will not linger any more upon the road, but follow our guide, who hurries forward along the intricate passages until he lands us at last in an irregularly shaped space, illuminated by a luminare, decorated with paintings, and bearing manifest tokens of having been once a great centre of devotion. There is the pillar to support the usual vessel of oil or more precious unguents to be burnt before the tomb of the martyr; and hard by is a gravestone let into the wall with the words C M , E .
The stone does not close one of the common graves such as are seen in the walls of the galleries or of the cubicula, neither is the grave an ordinary arcosolium The lower part of it, indeed, resembles an arcosolium inasmuch as it is large enough to contain three or four bodies, but there is no arch over it. The opening is rectangular, not circular, and yet there is no trace of any slab having been let into the wall to cover the top of the grave. It is probable, therefore, that a sarcophagus once filled the vacant space, and that the top of this
sarcophagus served as the mensa or altar, an arrangement of which other examples have been found.
But how came Pope Cornelius to be buried here, and not with his predecessors in the Papal Crypt? He was Pope, . . 250, between Fabian and Lucius, both of whom were buried, as we have seen, in that crypt. It is to be observed, however, that Cornelius is the only Pope, during the first three centuries, who bore the name of a noble Roman family; and many ancient epitaphs have been found in the area round this tomb, of persons who belonged to the same family. It is obvious, therefore, to conjecture that this sepulchre was the private property of some branch of the Gens Cornelia. The public Cemetery of St. Callixtus may have been closed at this time by order of the Government; but even without such a reason, it may have been the wish of the family that the Pope should not be separated in burial from the rest of his race. The same circumstance would account for the epitaph being written in Latin, not in Greek, for many of the old patrician families clung to the language of their forefathers long after the use of Greek had come into fashion; and this departure from the official language of the Church (for such, in fact, Greek really was at that time) is quite of a piece with the preference of the domestic to the official burial-place.
But whatever may be the true explanation of these circumstances, the fact is at least certain that Cornelius was buried here; and above and below the opening of his tomb are fragments, still adhering to the wall, of large slabs of marble, containing a few letters of what were once important inscriptions. The upper inscription was unquestionably the work of Damasus. The letters of the lower, though closely resembling the Damasine type, yet present a few points of difference—sufficient to warrant the conjecture of De Rossi that they were executed by the same hand, but with slight variations, in order to mark that it belonged to another series of monuments. We subjoin a copy of both inscriptions, in the form in which De Rossi believes them to have been originally written. In the first inscription the difference of type will distinguish the earlier half of each line, which is a conjectural restoration, from the latter half which still remains in situ; and in estimating the degree of probability of the
restorations, the reader should bear in mind two things: first, that the Damasine inscriptions were engraved with such mathematical precision that no emendations are admissible which would materially increase or diminish the number of letters in each line; and secondly, that whereas Damasus was in the habit of repeating himself very frequently in his epitaphs, several of De Rossi’s restorations are mere literal reproductions of some of his favourite forms of speech. Had the following epitaph been found in some ancient MS., and there attributed to Pope Damasus, we are confident that no critic would have seen reason to doubt its genuineness:—
ASPICE, DESCENSU EXSTRUCTO TENEBRISQUE FUGATIS,
CORNELI MONUMENTA VIDES TUMULUMQUE SACRATUM. HOC OPUS ÆGROTI DAMASI PRÆSTANTIA FECIT, ESSET UT ACCESSUS MELIOR, POPULISQUE PARATUM
AUXILIUM SANCTI, ET VALEAS SI FUNDERE PURO CORDE PRECES, DAMASUS MELIOR CONSURGERE POSSET, QUEM NON LUCIS AMOR, TENUIT MAGE CURA LABORIS.
“Behold, a new staircase having been made, and the darkness put to flight,
You see the monuments of Cornelius and his sacred tomb. This work the zeal of Damasus has accomplished, at a time when he was sick; That so the means of approach might be better, and the aid of the saint
Put more within the reach of the people; and that if you pour forth prayers
From a pure heart, Damasus may rise up in better health; Though it has not been love of life, but rather anxiety for work, that has retained him in this life.”
The second inscription De Rossi would restore as follows:—
SIRICIUS PERFECIT OPUS, CONCLUSIT ET ARCAM
MARMORE, CORNELI QUONIAM
PIA MEMBRA RETENTAT
—that is to say, he supposes that, Damasus having died, his successor Siricius completed the work that had been begun, and, furthermore, strengthened the wall which enclosed the tomb of St. Cornelius with this very thick slab of marble—a work which may have been rendered necessary by the alterations already made by Damasus. Of course, these restorations of the mutilated inscriptions must always remain more or less doubtful, for we fear there is no chance of any other fragments of the original ever coming to light. We publish them under the same reserve with which he himself proposes them, as at least approximations to the truth. He says that, without daring to affirm their literal correctness, there are certainly strong reasons for believing that they exactly reproduce the sense of the original.
This same tomb of St. Cornelius will supply us with an example of De Rossi’s power of happy conjecture, confirmed with absolute certainty by subsequent discoveries. He had often publicly expressed his confident expectation of finding at this tomb of St. Cornelius some memorial of his cotemporary, St. Cyprian. These two saints were martyred on the same day, though in different years; and their feasts were, therefore, always celebrated together, just as they are now, on the 16th of September, all the liturgical prayers for the day being common to both. Now, De Rossi had found in one of the old Itineraries, to whose accuracy of detail he had been greatly indebted, an extraordinary misstatement, viz., that the bodies of both these saints rested together in the same catacomb, whereas everybody knows that St. Cyprian was buried in Africa. He conjectured, therefore, that the pilgrim had been led into this blunder by something he had seen at the tomb of St. Cornelius. On its rediscovery, the cause of the error stands at once revealed. Immediately on the right hand side of the grave are two large figures of bishops painted on the wall, with a legend by the side of each, declaring them to be St. Cornelius and St. Cyprian.
On the other side of the tomb is another painting, executed in the same style, on the wall at the end of the gallery: two figures of
bishops, again designated by their proper names and titles. Only one of these can now be deciphered, ̅ ̅ ̅ ̅ ̅ ̅ ̅, i.e., Pope Sixtus II., of whose connection with this cemetery we have already heard so often. The other name began with an O, and was probably St. Optatus, an African bishop and martyr, whose body had been brought to Rome and buried in this cemetery.
These paintings are manifestly a late work: perhaps they were executed in the days of Leo III., . . 795-815, of whom it is recorded in the Liber Pontificalis, that “he renewed the Cemetery of Sts. Sixtus and Cornelius on the Appian Way;” and the legend which runs round them would have a special significance as the motto of one who had been almost miraculously delivered out of the hands of his enemies by the Emperor Charlemagne. It is taken from the 17th verse of the 58th Psalm: “Ego autem cantabo virtutem Tuam et exaltabo misericordiam Tuam quia factus es et susceptor meus.”... “I will sing Thy strength, and will extol Thy mercy, for Thou art become my support.” Of course, this had not been the earliest ornamentation of these walls. Even now, we can detect traces of a more ancient painting, and of graffiti upon it, underlying this later work. The graffiti are only the names of priests and deacons, who either came here to offer the holy sacrifice, or perhaps to take part in the translation of the relics: “Leo prb., Theodorus prb., Kiprianus Diaconus,” &c.
We are drawing very near to the end of our subterranean walk: indeed, the staircase which is to restore us to the upper air close to the very entrance of the vineyard is immediately behind us, as we stand contemplating the tomb of St. Cornelius. Nevertheless, if we are not too weary, nor our guide too impatient, we should do well to resist the temptation to escape, until we have first visited two small chambers which are in the immediate neighbourhood. They contain some of the most ancient specimens of painting to be found in the whole range of the Catacombs. The ceilings are divided into circles and other geometrical figures, and then the spaces are filled up with graceful arabesques, birds, and flowers, peacocks, and dancing genii. It was the sight of such paintings as these which led the Protestant writer quoted in a former chapter to express an opinion that, on first entering some of the decorated chambers in the
Catacombs, it is not easy to determine whether the work is Christian or Pagan. Here, indeed, the Good Shepherd in one centre and Daniel between two lions in the other soon solve the doubt; but all the other details and the excellence of their execution may well have suggested it. No one can doubt that the paintings belong to the very earliest period of Christian art, when the forms and traditions of the classical age had not yet died away.
In the first of the two chambers we are speaking of, there is nothing special to be seen besides the ceiling; but the second and more distant is more richly decorated. Here, two sepulchral chambers open one into the other: over the doorway which admits to the inner vault is represented the Baptism of our Lord by St. John: He is coming up out of the water and the dove is descending upon Him. On the wall opposite to the entrance is that fish carrying the basket of bread and wine that has been already described (page 81). On the wall to the left is a pail of milk standing on a kind of altar between two sheep, and we know from St. Irenæus and from some of the earliest and most authentic acts of the martyrs that milk was an accepted symbol of the Holy Eucharist. Opposite to this are doves and trees, which are often used as types of the souls of the blessed in Paradise. Thus, on one side we have the faithful on earth standing around the Divine food which prepares for heaven; and on the other, souls released from the prison of the body have flown away and are at rest, reposing amid the joys of another world; so that it would almost seem as though the same sequence of ideas presided over the decoration of these chambers, as was certainly present to the minds of those who designed the ornamentation of the sacramental chambers in the Cemetery of St. Callixtus (page 84).
And now at length we must conclude our visit to St. Callixtus. We fear that we have already enumerated more than can be seen with advantage during the course of a single visit; yet it is worth an effort to see it all, because it includes monuments which illustrate nearly every century of the period during which the Catacombs were used. It is for this reason that a visit to St. Callixtus is so singularly valuable, whether it be intended to take this cemetery as a sample of all, or only to use it as an introduction to others. Those who propose
to pursue the subject further would do well to visit next the Catacomb of SS. Nereus and Achilles, which lies at no great distance, off the Via Ardeatina; then the Cemetery of Pretextatus on the other side of the Via Appia; and finally, the Cœmeterium Ostrianum on the Via Nomentana. When these have been carefully examined, there will still remain many interesting monuments, of considerable historical importance, in other less famous cemeteries; but enough will have been seen to give an excellent general acquaintance with the main characteristics of Roma Sotterranea.
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